Project Summary Lyme disease caused by is the most prevalent human tick-borne disease in the United States. There is strong evidence that tick transmission of Lyme disease agents is enhanced by tick saliva proteins. Human Lyme disease is primarily associated with infectious bites of the blacklegged nymph ticks. However, the molecular identities of Borrelia burgdorferi-infected blacklegged nymph tick salivary proteins and their functional roles in transmission and promoting the Lyme disease agent infection of the host are not well defined. These data will be critical to understanding molecular systems that control tick transmission of the Lyme disease agents, which in turn will be important to developing the highly desired tick-antigen based transmission blocking vaccine to prevent Lyme disease in humans. Therefore, the broad goals of this application are to utilize to identify and define functions of tick saliva proteins that are associated with transmission of the Lyme disease agent. The rationale for this approach is to identify tick saliva proteins that are induced or are differentially enhanced in response to infection of nymphs. We plan to identify Borrelia burgdorferi infection responsive tick saliva proteins over the 12-72 h feeding period when the Lyme disease agent is expected to be transmitted. The novelty of our data is that we will determine both molecular identities and the timelines at which Bb infection associated TSPs are injected into the host during the critical 12-72 h tick feeding period. We will juxtapose these data against published tick feeding timelines of Borrelia burgdorferi transmission to identify early stage tick feeding tick saliva proteins that precede major Borrelia burgdorferi transmission events, and middle/late stage tick feeding tick saliva proteins that coincide with major transmission events. In the short-term, we will undertake systematic RNAi silencing analysis to determine which of the two clusters of tick saliva proteins are important to transmission and Borrelia burgdorferi infection. Data from this application will serve as the foundation for our long-term goals to develop tick-antigen based Lyme disease agent transmission blocking vaccines. We will test the hypothesis that Bb infection of the I. scapularis nymph tick will induce expression of tick saliva proteins that promote transmission and Borrelia burgdorferi infection of the host, which if disrupted will protect the host against Lyme disease. The goals of this research will be accomplished in two specific aims: (i) to identify tick saliva proteins associated with transmission of Borrelia burgdorferi by Ixodes scapularis nymph ticks, and (ii) to validate roles of Borrelia burgdorferi infection associated tick saliva proteins in Borrelia burgdorferi transmission by ticks.